The biological roles of nitric oxide (NO) have led chemists and molecular biologists to seek cellular imaging agents responsive to this species. The creation of such agents derives from the pivotal role of NO in vasodilation as an endothelial-derived relaxing factor (EDRF), and its function as a platelet aggregation inhibitor, neurotransmitter, antimicrobial agent, and due to its antitumor activity in cardiovascular, nervous, and immune systems. Although a variety of quantification techniques have been developed, fluorescence techniques are the most desirable because of their sensitivity, and high spatiotemporal resolution when combined with microscopy. Consequently, a number of fluorescent NO probes are available, but each is hampered by certain selectivity and/or synthetic limitations.
Currently, the most common approach for NO detection involves the use of ortho-diamino aromatics under aerobic conditions, which reacts with NO+ equivalent, presumably N2O3 to furnish fluorescent triazole derivatives. Turn-on fluorescence signals are achieved due to suspension of photoinduced electron transfer (PET). Examples using fluoresceins (such as DAF-2 DA), anthraquinones, rhodamines (such as DAR-4M AM), BODIPYs, and cyanines are documented. Such probes are among the current state of the art, yet severe limitations exist. First of all, in the presence of H2O2/peroxidase, OONO−, OH., NO2., and CO3.−, the intrinsically electron rich diaminobenzene moiety is easily oxidized to an arylaminyl radical, which combines with NO and leads to triazoles. Second, dehydroascorbic acid (DHA) condenses with ortho-diamino aromatics and turns on the fluorescence of such probes. It was reported that 1 mM DHA yielded a fluorescence signal with the commercial NO probes DAF-2 DA, DAR-4M AM, comparable to 300 nM and 100 μM of NO respectively. Third, benzotriazoles are pH sensitive (pKa's≈6.69) near neutral pH. The pH sensitivity can be solved by methylation of one of the amines, however the reactivity of the probe toward DHA was undesirably enhanced.
The aforementioned limitations complicate NO detection using ortho-diamines. Hence, a series of metal ligand complexes for NO detection are also currently under development. For example, CuII(FL5), displays a fluorescence enhancement upon exposure to NO and can be used as a cellular imaging agent. However, given a dissociation constant (Kd) of 1.5 μM and the presence other metal ions in physiological conditions, it is a concern that the complex will release cytotoxic Cu2+. Complexes with lower Kd's were reported, though with decreased reactivity toward NO.
Most recently, single-walled carbon nanotubes (SWCN) wrapped with 3,4-diaminophenyl-functionalized dextrans were used for in vitro or in vivo studies. The NIR fluorescence of the SWCN is bleached by several reactive oxygen/nitrogen species, but at least NO does so more than others.